(CANCER RESEARCH 52. 3409-3417. June 15, 1992] Resistance to /V-Benzyladriamycin-14-valerate in Mouse J774.2 Cells: P-Glycoprotein Expression without Reduced jV-Benzyladriamycin- 14-va lenite Accumulation1 Leonard Lothstein,2 Trevor W. Sweatman, Michael E. Dockter, and Mervyn Israel Departments of Pharmacology [L. L., T. W. S., M. I.] and Medicine [M. E. D.J, College of Medicine, and Cancer Center [L. L., T. W. S., M. 1.], University of Tennessee- Memphis, Memphis, Tennessee 38163 ABSTRACT Ar-Benzyladriamycin-14-valerate (AD 198) ¡s a highly lipophilic ana logue of Adriamycin with novel cytotoxic mechanisms, greater in vivo antitumor activity, and the ability to circumvent multidrug resistance due to P-glycoprotein-mediated drug efflux or decreased topoisomerase II activity. To identify the mechanism(s) which may confer AD 198 resist ance, J774.2 mouse macrophage-like cells were selected for growth in cytotoxic levels of AD 198 (AD 198"). AD 198" cells exhibited over- expression of the mtlrlb (P-glycoprotein) gene, cross-resistance to Adri- amycin and vinblastine, and potentiation of drug cytotoxicity by verapa- mil. However, net intracellular accumulation of AD 198 in AD 198" cells was unchanged compared to parental cells, while Adriamycin and vin blastine accumulations were reduced 40% and 95%, respectively. AD 198 was localized in the perinuclear region of the cytoplasm in both parental and AD 198" cells, with additional vesicular compartmentalization in AD 198R cells. Verapamil-induced reversal of AD 198 resistance coincided with some drug redistribution from cytoplasmic vesicles, but without redistribution of AD 198 into the nucleus. These results suggest that AD 198 resistance was not conferred through a P-glycoprotein-mediated reduction in intracellular drug accumulation but through other cyto plasmic mechanisms, including, but not limited to, drug compartmental ization. INTRODUCTION The anthracycline antibiotic ADR1 is an effective cancer chemotherapeutic agent that has been widely used against a variety of human malignancies (1). Concomitant with its anti- tumor activity, ADR can produce adverse systemic effects, including acute myelosuppression, cumulative cardiotoxicity, and gastrointestinal toxicity (2). At the cellular level, in both cultured mammalian cells and primary tumor cells, ADR can select for multiple mechanisms of drug resistance that decrease its chemotherapeutic efficacy. These mechanisms include P-gp- mediated MDR, characterized by the energy-dependent trans port of drugs from the cell (3), and multidrug resistance con ferred by decreased topoisomerase II activity, resulting in de creased ADR-induced DNA strand scission (4-6). Among the potential avenues of circumvention of systemic toxicity and cellular drug resistance is the development of semisynthetic Received 3/11/91; accepted 4/3/92. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported in part by American Cancer Society Grants IN-176-A and JFRA- 287 (L. L.), Pharmaceutical Manufacturers Association Foundation Research Starter Grant (L. L.), and NIH-National Cancer Institute Research Grants CA44890 (T. W. S.), CA37082 (M. I.), and CA37209 (M. I.). 2To whom requests for reprints should be addressed, at Department of Pharmacology, University of Tennessee College of Medicine, 874 Union Avenue, Memphis, TN 38163. 3The abbreviations used are: ADR, Adriamycin hydrochloride (doxorubicin); AD 198, /V-benzyladriamycin-14-valerate; AD 288, A'-benzyladriamycin; DMSO, dimethyl sulfoxide; MDR, multidrug resistance (resistant); P-gp, P-glycoprotein; VBL, vinblastine sulfate; VRP, (±)verapamil; cDNA, complementary DNA; HPLC, high-performance liquid chromatography; IC50,drug concentration inhib iting growth by 50% after 72 h of continuous exposure; TBS, 20 mm Tris-HCl, pH 7.5, 0.5 M NaCl; TTBS, 0.2% Tween 20 in TBS; PBS, phosphate-buffered saline. ADR analogues which demonstrate greater tumor-specific tox icity and less susceptibility to various forms of resistance. One such highly hydrophobic analogue, AD 198, exhibits a variety of mechanistic differences compared with ADR, including weaker binding to purified DNA, preferential inhibition of RNA versus DNA synthesis, irreversible G2/M blockade, pro nounced membrane lytic activity, and a lack of inhibition of purified mammalian topoisomerase II despite significant levels of protein-associated DNA strand breaks in alkaline elution assays (2, 7-9). When compared with ADR, AD 198 demon strates enhanced cytotoxicity against cultured murine and hu man tumor cells and the ability to circumvent MDR in P388 and LI210 leukemic cells and B16-BL6 melanoma cells, and both MDR and resistance due to altered topoisomerase II activity in variant CCRF-CEM leukemic cells (10, 11). Despite the high degree of toxicity seen in vitro, AD 198 exhibits limited efficacy against transplanted MDR LI210 cells in vivo. This observation suggests that resistance to AD 198 may be con ferred either systemically through enhanced drug metabolism or pharmacologie sanctuary of the neoplasia or through cellular resistance (10). In an effort to identify the mechanism(s) by which mamma lian cells may acquire resistance to AD 198, the selection of resistant variants of the mouse macrophage-like cell line J774.2 were attempted by continuous exposure to increasing concen trations of AD 198. J774.2 cells are amenable to the rapid selection of drug-resistant variants, a number of which have been extensively analyzed (12-17). Selection of variant J774.2 cells would represent the first reported selection of myeloid cells for resistance to AD 198. In this report, it is shown that (a) AD 198-resistant variants of J774.2 (AD 198R) can be rapidly selected; (b) AD 198Rcells exhibit several characteristics of MDR, including P-gp overexpression, cross-resistance to other drugs, and attenuation of resistance by the calcium chan nel blocker VRP; and (c) unlike cross-resistance to ADR and VBL, AD 198 resistance is not associated with reduced net AD 198 accumulation. MATERIALS AND METHODS Materials. AD 198 (Fig. 1), [14C]AD 198 (3.8 mCi/mmol), and [I4C] ADR (5.05 mCi/mmol) were prepared as described previously (8, 11). VBL and VRP were obtained from Sigma Chemical Co. (St. Louis, MO). ADR was a generous gift from Farmitalia Carlo Erba (Milan, Italy). [3H]VBL (11.2 Ci/mmol) was obtained from Amersham Corp. (Arlington Heights, IL), and [-"P]dCTP (3000 Ci/mmol) was from NEN/DuPont (Boston, MA). Drug-sensitive J774.2 cells, VBL-resist- ant J774.2 cells (13, 17), and mdr cDNA probes (14) were generously supplied by Dr. Susan Honvitz (Albert Einstein College of Medicine, Bronx, NY). Cell Culture. The propagation J774.2 cells and the selection of VBL- resistant J7.V3-0.04 cells have been described previously (13, 14, 18). AD 198R variants were selected by stepwise, continuous exposure of J774.2 cells to increasing concentrations of AD 198, beginning with 50 3409 Research. on February 11, 2016. © 1992 American Association for Cancer cancerres.aacrjournals.org Downloaded from